US4529708A - Assay for the determination of creatinine - Google Patents

Assay for the determination of creatinine Download PDF

Info

Publication number
US4529708A
US4529708A US06/482,753 US48275383A US4529708A US 4529708 A US4529708 A US 4529708A US 48275383 A US48275383 A US 48275383A US 4529708 A US4529708 A US 4529708A
Authority
US
United States
Prior art keywords
creatinine
reagent
protein
picrate
bilirubin
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Expired - Fee Related
Application number
US06/482,753
Inventor
Thomas W. Stephens
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
INDIANA DEVELOPMENT FINANCE AUTHORITY
American Monitor Corp
Original Assignee
American Monitor Corp
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by American Monitor Corp filed Critical American Monitor Corp
Priority to US06/482,753 priority Critical patent/US4529708A/en
Assigned to AMERICAN MONITOR CORPORATION reassignment AMERICAN MONITOR CORPORATION ASSIGNMENT OF ASSIGNORS INTEREST. Assignors: STEPHENS, THOMAS W.
Priority to EP19840901699 priority patent/EP0143805A4/en
Priority to PCT/US1984/000532 priority patent/WO1984003948A1/en
Application granted granted Critical
Publication of US4529708A publication Critical patent/US4529708A/en
Assigned to NEUBERGER AND BERMAN (LENDER), 522 FIFTH AVENUE, NEW YORK NEW YORK 10036 A NEW YORK LIMITED PARTNERSHIP reassignment NEUBERGER AND BERMAN (LENDER), 522 FIFTH AVENUE, NEW YORK NEW YORK 10036 A NEW YORK LIMITED PARTNERSHIP SECURITY INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: AMERICAN MONITOR CORPORATION
Assigned to AMERICAN MONITOR PARTNERS reassignment AMERICAN MONITOR PARTNERS SECURITY INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: AM DIAGNOSTICS, A CORP. OF IN
Assigned to FOOTHILL CAPITAL CORPORATION, A CA. CORP. reassignment FOOTHILL CAPITAL CORPORATION, A CA. CORP. SECURITY INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: AM DIAGNOSTICS, A CORP. OF IN
Assigned to INDIANA DEVELOPMENT FINANCE AUTHORITY reassignment INDIANA DEVELOPMENT FINANCE AUTHORITY ASSIGNMENT OF ASSIGNORS INTEREST. Assignors: AM DIAGNOSTICS, INC.
Anticipated expiration legal-status Critical
Expired - Fee Related legal-status Critical Current

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/70—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving creatine or creatinine
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
    • Y10T436/00—Chemistry: analytical and immunological testing
    • Y10T436/14—Heterocyclic carbon compound [i.e., O, S, N, Se, Te, as only ring hetero atom]
    • Y10T436/145555—Hetero-N
    • Y10T436/147777—Plural nitrogen in the same ring [e.g., barbituates, creatinine, etc.]
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
    • Y10T436/00—Chemistry: analytical and immunological testing
    • Y10T436/25—Chemistry: analytical and immunological testing including sample preparation
    • Y10T436/25125—Digestion or removing interfering materials

Definitions

  • This invention relates to an improved assay method and reagent composition for determining creatinine levels in body fluids, and more particularly to a colorimetric method which is performed directly on serum and other fluids without deproteinization.
  • Creatinine is synthesized in the body at a fairly constant rate from creatine, which is produced during muscle contractions from creatine phosphate. Creatinine in the blood is then removed by filtration through the glomeruli of the kidney for excretion in the urine. Since the excretion of creatinine in healthy individuals is independent of diet and thus relatively constant, the creatinine clearance test is one of the most sensitive tests for measuring glomerular filtration rate, and the concentration of creatinine in the serum depends almost entirely upon its rate of excretion by the kidney. In kidney disease, therefore, creatinine levels in the blood are elevated, while the creatinine clearance, or urine levels, are diminished.
  • the Jaffe reaction achieves a measurable result, it has long been recognized that the reaction suffers a significant drawback in its lack of specificity for measuring creatinine in serum and urine. More particularly, a number of other substances which are normally present in serum and urine also form a red color with picrate or otherwise interfere with the reaction, thereby causing inaccurate and misleading results. Most of these substances cause a positive interference by increasing the amount of color formation. Such substances include, for example, protein, glucose, ascorbate, acetoacetate, pyruvate and other alpha-keto acids. On the other hand, bilirubin in the presence of protein causes a negative interference, or falsely low results.
  • pretreatment methods are cumbersome and time-consuming.
  • pretreatment methods have a particular disadvantage in that they do not easily lend themselves to automation, which has become a virtual necessity in meeting the demands of today's clinical laboratories.
  • pretreatment in many cases may solve only a portion of the nonspecificity problems or may even create additional specificity problems.
  • a more particular object of the present invention is to provide a method which employs a kinetic measurement of the reaction of creatinine in an alkaline picrate reagent.
  • a further object is to provide a reagent and assay method in which there is negligible interference from bilirubin.
  • Yet a further object is to provide a method which is readily adaptable for use with the variety of automated and semi-automated laboratory instrumentation in current use today.
  • Another object is to provide a reagent composition with enhanced stability.
  • the preferred iron chelator is ethylenediaminetetraacetic acid (EDTA); however, N-hydroxyethylethylenediaminetetraacetic acid (HEDTA) and ethylene glycol-bis( ⁇ -amino-ethyl ether)-N,N'-tetraacetic acid (EGTA) have also been found to be useful.
  • EDTA ethylenediaminetetraacetic acid
  • HEDTA N-hydroxyethylethylenediaminetetraacetic acid
  • EGTA ethylene glycol-bis( ⁇ -amino-ethyl ether)-N,N'-tetraacetic acid
  • the buffered alkaline picrate solution is preferably of the nature as described and set forth in the aforementioned Denney and Long patent, i.e., dimethylsulfoxide is also included in the reagent to suppress protein interference.
  • surface active agents known to be useful in suppressing turbidity may also be included.
  • EDTA is added to the reagent in the preferred amount of at least about 0.5 millimoles per liter. This quantity of EDTA has been found to be sufficient to adequately suppress negative bilirubin interference in specimens containing as much as 20 milligrams per deciliter (mg/dl) of bilirubin. (The minimum concentration of bilirubin necessary to visibly discolor serum is approximately 3 mg/dl, while normal bilirubin levels are generally less than 1 mg/dl.)
  • a sample of serum, urine, or other fluid is combined with the reagent.
  • the rate of formation of the colored picramate complex is measured photometrically at a wavelength between about 480 nm and 530 nm.
  • Calibration of the assay is accomplished by similarly treating an equal volume of a standard or reference material containing a known amount of creatinine.
  • kits pre-formulated compositions, or reagents, which are generally known as "test kits", and which are available on a commercial basis from various manufacturers.
  • a kit may contain one or more pre-formulated reagents and appropriate calibration and quality control materials, or the kit may be in the form of one or more pre-formulated reagents packaged individually or in bulk form for a specific intended use.
  • the test kit contain two separate, pre-formulated reagents, one containing picrate and the other containing an alkaline buffer. Either or both reagents may also contain additional desired ingredients such as surfactants, antimicrobial agents, and others that will be known to those skilled in the art. With respect to the present invention, the EDTA may be added to either reagent, but solubility has been found to be better if included in the alkaline buffer.
  • An alkaline buffer reagent is prepared by combining the following substances in the amount indicated in sufficient deionized water to make a total volume of one liter:
  • the resulting solution should be approximately 0.4 N sodium hydroxide.
  • a picrate reagent is prepared by combining the following substances in the amount indicated in sufficient deionized water to make a total volume of one liter:
  • Neodol is a trademark of Shell Chemical Company, Houston, Tex. Neodol 25-3S is an ethoxysulfate surfactant.
  • a diagnostic reagent for the determination of creatinine is then prepared by combining equal quantities of the alkaline buffer and the picrate reagents.
  • the final reagent thus prepared has been found to retain its utility for at least thirty (30) days, while the alkaline buffer and picrate reagents stored separately are expected to retain their utility for at least two years.
  • an amount of the sample to be assayed and an amount of the reagent are combined in the preferred ratio of 1 part sample to 20 parts of the reagent.
  • the rate of increase in absorbance at 510 nm is then measured and compared to the rate observed with a calibrator of known creatinine concentration.
  • the KDA® analyzer has been used in carrying out this embodiment of the present invention. (KDA is a registered trademark of American Monitor Corporation.)
  • An alkaline buffer reagent is prepared by combining the following substances in the amount indicated in sufficient deionized water to make a total volume of one liter:
  • the resulting solution should be approximately 0.18 N sodium hydroxide.
  • a picrate reagent is prepared as described above in Example 1.
  • a diagnostic reagent for the determination of creatinine is then prepared by combining equal quantities of the alkaline buffer and the picrate reagents.
  • the final reagent thus prepared has been found to retain its utility for at least thirty (30) days, while the alkaline buffer and picrate reagents stored separately are expected to retain their utility for at least two years.
  • EDTA concentration of 0.5 millimoles per liter of prepared diagnostic reagent is presented in the preferred embodiments set forth herein, amounts up to 5.0 millimolar or greater may be used in the practice of the present invention.
  • bind iron substances known to be useful in the practice of the present invention.
  • ethyleneglycol-bis( ⁇ -amino-ethyl ether)-N,N'-tetraacetic acid (EGTA), and N-hydroxyethylethylenediaminetetraacetic acid (HEDTA) have been found to also significantly suppress or eliminate bilirubin interference.
  • optical measurements of the reaction need not necessarily be limited to absorbance-measuring techniques. It is conceivable that transmittance-measuring and even reflectance-measuring techniques may also be employed. Other modifications and perturbations to the foregoing embodiments will be apparent to those skilled in the art and are not to be considered beyond the scope of the novel concepts of the present invention.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Chemical & Material Sciences (AREA)
  • Hematology (AREA)
  • Immunology (AREA)
  • Urology & Nephrology (AREA)
  • Cell Biology (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

Abstract

An improved colorimetric assay method and reagent composition for the quantitative determination of creatinine in biological fluid specimens in the presence of protein, in which the specimen is reacted directly with an alkaline picrate reagent composition which contains an iron chelator, preferably ethylenediaminetetraacetic acid, thereby significantly suppressing interference from bilirubin and improving reagent stability.

Description

BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates to an improved assay method and reagent composition for determining creatinine levels in body fluids, and more particularly to a colorimetric method which is performed directly on serum and other fluids without deproteinization.
2. Nature and Significance of Accurate Creatinine Determinations
It has been recognized for almost a century that the measurement of creatinine is of vital medical significance, and even today, the most widely used kidney function test in clinical practice is the estimation of glomerular filtration rate by measurement of the creatinine level in serum and urine specimens. Tests or assays that measure such creatinine levels are major criteria in determining whether kidney disease is present and, if present, the extent to which it is present.
Creatinine is synthesized in the body at a fairly constant rate from creatine, which is produced during muscle contractions from creatine phosphate. Creatinine in the blood is then removed by filtration through the glomeruli of the kidney for excretion in the urine. Since the excretion of creatinine in healthy individuals is independent of diet and thus relatively constant, the creatinine clearance test is one of the most sensitive tests for measuring glomerular filtration rate, and the concentration of creatinine in the serum depends almost entirely upon its rate of excretion by the kidney. In kidney disease, therefore, creatinine levels in the blood are elevated, while the creatinine clearance, or urine levels, are diminished.
3. Discussion of the Prior Art
Prior art methods date as far back as 1886 when Jaffe (Z. Physiol. Chem. 10:391, 1886) described the formation of a red color when creatinine was reacted with picrate in an alkaline solution. Today, this classic Jaffe reaction remains the basis for a majority of the current creatinine assay methods.
Although the Jaffe reaction achieves a measurable result, it has long been recognized that the reaction suffers a significant drawback in its lack of specificity for measuring creatinine in serum and urine. More particularly, a number of other substances which are normally present in serum and urine also form a red color with picrate or otherwise interfere with the reaction, thereby causing inaccurate and misleading results. Most of these substances cause a positive interference by increasing the amount of color formation. Such substances include, for example, protein, glucose, ascorbate, acetoacetate, pyruvate and other alpha-keto acids. On the other hand, bilirubin in the presence of protein causes a negative interference, or falsely low results.
To overcome the problems of non-specific color formation, numerous methods have been developed which remove the interfering substance by some type of sample pretreatment, the most commonly performed pretreatment methods being protein precipitation and removal by filtration, treatment with Lloyd's reagent, or removal of protein by dialysis through a membrane.
However, such pretreatment methods are cumbersome and time-consuming. Furthermore, with the exception of protein removal by dialysis, pretreatment methods have a particular disadvantage in that they do not easily lend themselves to automation, which has become a virtual necessity in meeting the demands of today's clinical laboratories. Additionally, pretreatment in many cases may solve only a portion of the nonspecificity problems or may even create additional specificity problems.
In 1971, Bartels and Bohmer (Clin. Chem. Acta 32:81, 1971) and Cook (Clin. Chim. Acta 32:485, 1971) proposed the measurement of creatinine in the presence of protein by measuring the rate of color development of the classic Jaffe reaction, i.e., kinetic measurement. Although many different means have been attempted in past years to improve the specificity of the Jaffe reaction, the most popular means of improving specificity of creatinine assays has been the use of kinetic methods based on the Jaffe reaction. Denney and Long (U.S. Pat. No. 4,111,657, issued Sept. 5, 1978) suppressed protein interference in a kinetic creatinine assay by the incorporation of dimethylsulfoxide in an alkaline picrate reagent. Although their method achieved several important advantages over earlier attempts, their modifications left unsolved the problem of the negative interference from bilirubin present in serum specimens, i.e. icteric samples. Many investigators have recently studied and attempted to correct for this negative interference, which appears to exist in virtually all of today's popularly used creatinine assay methods which are carried out in the presence of protein.
Two investigators, Tagesson and Rebel (Clin. Chem. 26:520, 1980), quite apart from attempting to solve bilirubin interference, attempted to resolve a problem of drift (a continuous gradual increase in results) in certain continuous flow creatinine methods. (Continuous flow methods rely upon the separation of protein from the specimens by use of a semi-permeable membrane.) Tagesson and Rebel postulated that calcium was precipitating in the cuvette in which optical density was measured and added EDTA to prevent the precipitation of calcium and thus reduce the drift causing increasing creatinine values. The present invention also incorporates EDTA, however achieving benefits quite in contrast to those of Tagesson and Rebel. In fact, the benefits of the present invention would be quite unnoticed and unappreciated in continuous flow methods since protein, and substances which are bound to protein, including bilirubin, are separated and removed from the samples being assayed.
SUMMARY OF THE INVENTION
Accordingly, it is therefore a general object of the present invention to provide a novel reagent composition and assay method for the determination of creatinine in fluid samples in the presence of protein. A more particular object of the present invention is to provide a method which employs a kinetic measurement of the reaction of creatinine in an alkaline picrate reagent. A further object is to provide a reagent and assay method in which there is negligible interference from bilirubin. Yet a further object is to provide a method which is readily adaptable for use with the variety of automated and semi-automated laboratory instrumentation in current use today. Another object is to provide a reagent composition with enhanced stability. These and other objects, features, and advantages will be apparent from the following description of the presently preferred embodiments of the present invention.
It has been discovered that when an agent which binds or chelates iron is included in a buffered alkaline picrate solution, the objects and goals of the present invention are achieved. The preferred iron chelator is ethylenediaminetetraacetic acid (EDTA); however, N-hydroxyethylethylenediaminetetraacetic acid (HEDTA) and ethylene glycol-bis(β-amino-ethyl ether)-N,N'-tetraacetic acid (EGTA) have also been found to be useful. The buffered alkaline picrate solution is preferably of the nature as described and set forth in the aforementioned Denney and Long patent, i.e., dimethylsulfoxide is also included in the reagent to suppress protein interference. Optionally, surface active agents known to be useful in suppressing turbidity may also be included.
EDTA is added to the reagent in the preferred amount of at least about 0.5 millimoles per liter. This quantity of EDTA has been found to be sufficient to adequately suppress negative bilirubin interference in specimens containing as much as 20 milligrams per deciliter (mg/dl) of bilirubin. (The minimum concentration of bilirubin necessary to visibly discolor serum is approximately 3 mg/dl, while normal bilirubin levels are generally less than 1 mg/dl.)
To use the reagent composition of the present invention to perform an assay for creatinine, a sample of serum, urine, or other fluid is combined with the reagent. The rate of formation of the colored picramate complex is measured photometrically at a wavelength between about 480 nm and 530 nm. Calibration of the assay is accomplished by similarly treating an equal volume of a standard or reference material containing a known amount of creatinine.
Although the present invention can be practiced by additions of the desired ingredients to the sample individually or in the form of one or more reagent combinations, it is customary and convenient for laboratory and other analytical personnel to use pre-formulated compositions, or reagents, which are generally known as "test kits", and which are available on a commercial basis from various manufacturers. A kit may contain one or more pre-formulated reagents and appropriate calibration and quality control materials, or the kit may be in the form of one or more pre-formulated reagents packaged individually or in bulk form for a specific intended use.
With respect to the present invention, it is preferable that the test kit contain two separate, pre-formulated reagents, one containing picrate and the other containing an alkaline buffer. Either or both reagents may also contain additional desired ingredients such as surfactants, antimicrobial agents, and others that will be known to those skilled in the art. With respect to the present invention, the EDTA may be added to either reagent, but solubility has been found to be better if included in the alkaline buffer.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
The specific embodiments of the present invention detailed herein are provided to enable an analyst skilled in the art to understand and make reagent compositions and to perform an assay according to the novel concepts and achievements of the present invention.
EXAMPLE 1 Reagent Preparation
An alkaline buffer reagent is prepared by combining the following substances in the amount indicated in sufficient deionized water to make a total volume of one liter:
20.5 milliliters sodium hydroxide,
50% solution
416 milligrams EDTA, tetrasodium
The resulting solution should be approximately 0.4 N sodium hydroxide.
A picrate reagent is prepared by combining the following substances in the amount indicated in sufficient deionized water to make a total volume of one liter:
4.2 grams picric acid
219 grams dimethylsulfoxide
4 milliliters® 25-3S
(Neodol is a trademark of Shell Chemical Company, Houston, Tex. Neodol 25-3S is an ethoxysulfate surfactant.)
A diagnostic reagent for the determination of creatinine is then prepared by combining equal quantities of the alkaline buffer and the picrate reagents. The final reagent thus prepared has been found to retain its utility for at least thirty (30) days, while the alkaline buffer and picrate reagents stored separately are expected to retain their utility for at least two years.
EXAMPLE 2 Determination of Creatinine
To perform an assay for creatinine, 4.0 milliliters of the reagent and 150 microliters of the sample to be assayed are mixed together. The rate of increase in absorbance (A) of the reaction mixture is then measured optically at a wavelength of about 510 nm. For calibration, standard or reference material containing a known amount of creatinine is similarly treated.
To calculate the amount of creatinine in the sample, divide the change in absorbance at 510 nm per minute (ΔA510) of the assayed sample by the ΔA510 of the calibrator, then multiply the result thus obtained by the known amount of creatinine in the calibrator.
EXAMPLE 3 Determination of Creatinine
Using an automated chemical analyzer capable of performing mathematical comparisons involving rate reactions, an amount of the sample to be assayed and an amount of the reagent are combined in the preferred ratio of 1 part sample to 20 parts of the reagent. The rate of increase in absorbance at 510 nm is then measured and compared to the rate observed with a calibrator of known creatinine concentration. The KDA® analyzer has been used in carrying out this embodiment of the present invention. (KDA is a registered trademark of American Monitor Corporation.)
EXAMPLE 4 Reagent Preparation
An alkaline buffer reagent is prepared by combining the following substances in the amount indicated in sufficient deionized water to make a total volume of one liter:
9.2 milliliters sodium hydroxide, 50% solution
416 milligrams EDTA, tetrasodium
The resulting solution should be approximately 0.18 N sodium hydroxide.
A picrate reagent is prepared as described above in Example 1.
A diagnostic reagent for the determination of creatinine is then prepared by combining equal quantities of the alkaline buffer and the picrate reagents. The final reagent thus prepared has been found to retain its utility for at least thirty (30) days, while the alkaline buffer and picrate reagents stored separately are expected to retain their utility for at least two years.
EXAMPLE 5 Determination of Creatinine
Using an automated chemical analyzer capable of performing mathematical comparisons involving rate reactions, 40 microliters of the sample to be assayed are combined with 1.0 milliliter of the reagent, or any combination that will provide a preferred ratio of 1 part sample to 25 parts reagent. The rate of increase in color at 500 nm is then measured and compared to the rate observed with a calibrator of known creatinine concentration. The Parallel® analytical system has been used in carrying out this embodiment of the present invention. (Parallel is a registered trademark of American Monitor Corporation.)
Although a final EDTA concentration of 0.5 millimoles per liter of prepared diagnostic reagent is presented in the preferred embodiments set forth herein, amounts up to 5.0 millimolar or greater may be used in the practice of the present invention. The beneficial effects derived from additional amounts of EDTA, however, do not appear to increase proportionately, and 0.5 millimolar has been found to be an adequate concentration to meet the requirements of most clinical situations.
Other substances known to bind iron have also been found to be useful in the practice of the present invention. For example, 0.5 millimolar quantities of ethyleneglycol-bis(β-amino-ethyl ether)-N,N'-tetraacetic acid (EGTA), and N-hydroxyethylethylenediaminetetraacetic acid (HEDTA) have been found to also significantly suppress or eliminate bilirubin interference.
Furthermore, optical measurements of the reaction need not necessarily be limited to absorbance-measuring techniques. It is conceivable that transmittance-measuring and even reflectance-measuring techniques may also be employed. Other modifications and perturbations to the foregoing embodiments will be apparent to those skilled in the art and are not to be considered beyond the scope of the novel concepts of the present invention.

Claims (3)

What is claimed is:
1. A method for the determination of creatinine in a protein-containing fluid, comprising the steps of:
(a) combining a sample of said protein-containing fluid with a reagent to form a reaction mixture, said reagent comprising:
(i) an alkalinizing agent,
(ii) picrate, and
(iii) a substance which binds iron in an amount sufficient to suppress interference from bilirubin; and
(b) optically measuring the change in said reaction mixture.
2. A method as recited in claim 1, wherein the substance which binds iron is selected from the group consisting of ethylenediaminetetraacetic acid (EDTA), N-hydroxyethylethylenediaminetetraacetic acid (HEDTA), and ethyleneglycol-bis (β-amino-ethyl ether)-N,N'-tetraacetic acid (EGTA).
3. A method as recited in claim 2, wherein the amount of the substance which binds iron is at least about 0.5 millimoles per liter.
US06/482,753 1983-04-07 1983-04-07 Assay for the determination of creatinine Expired - Fee Related US4529708A (en)

Priority Applications (3)

Application Number Priority Date Filing Date Title
US06/482,753 US4529708A (en) 1983-04-07 1983-04-07 Assay for the determination of creatinine
EP19840901699 EP0143805A4 (en) 1983-04-07 1984-04-05 Improved reagent composition and assay for the determination of creatinine.
PCT/US1984/000532 WO1984003948A1 (en) 1983-04-07 1984-04-05 Improved reagent composition and assay for the determination of creatinine

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
US06/482,753 US4529708A (en) 1983-04-07 1983-04-07 Assay for the determination of creatinine

Publications (1)

Publication Number Publication Date
US4529708A true US4529708A (en) 1985-07-16

Family

ID=23917320

Family Applications (1)

Application Number Title Priority Date Filing Date
US06/482,753 Expired - Fee Related US4529708A (en) 1983-04-07 1983-04-07 Assay for the determination of creatinine

Country Status (3)

Country Link
US (1) US4529708A (en)
EP (1) EP0143805A4 (en)
WO (1) WO1984003948A1 (en)

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4818703A (en) * 1985-10-23 1989-04-04 Pizzolante John M Stabilized alkaline picrate reagent for jaffe creatinine determination
US4908676A (en) * 1987-12-18 1990-03-13 Bio-Recovery Systems, Inc. Sensors for dissolved substances in fluids
US5071772A (en) * 1988-02-08 1991-12-10 Raffaele Beli Device for carrying out extemporaneous quantitative diagnostic tests on whole blood
WO1996003648A1 (en) * 1994-07-25 1996-02-08 Professional Clinic Laboratories, Inc. Method of determining renal clearances
US5602038A (en) * 1994-07-25 1997-02-11 Private Clinic Laboratories, Inc. Method of determining renal clearances
US20040023364A1 (en) * 1999-01-15 2004-02-05 Mitchell Owens Lateral flow test strip
US20070031914A1 (en) * 2005-08-05 2007-02-08 Wei Zhu Devices for analyte assays and methods of use
US20190187146A1 (en) * 2014-02-28 2019-06-20 Nitto Denko Corporation Urinalysis Device And Dry Reagent For Quantitative Urinalysis

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3894843A (en) * 1974-03-04 1975-07-15 Coulter Diagnostics Inc Determination of serum creatinine
US4111657A (en) * 1977-01-21 1978-09-05 American Monitor Corporation Creatinine assay and reagent system
JPS5529718A (en) * 1978-08-23 1980-03-03 Hitachi Ltd Creatinine analysis method

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3894843A (en) * 1974-03-04 1975-07-15 Coulter Diagnostics Inc Determination of serum creatinine
US4111657A (en) * 1977-01-21 1978-09-05 American Monitor Corporation Creatinine assay and reagent system
JPS5529718A (en) * 1978-08-23 1980-03-03 Hitachi Ltd Creatinine analysis method

Non-Patent Citations (9)

* Cited by examiner, † Cited by third party
Title
Daugherty et al., Clinical Chemistry, vol. 24, No. 2, 1978, pp. 392 393. *
Daugherty et al., Clinical Chemistry, vol. 24, No. 2, 1978, pp. 392-393.
Lolekha et al., Clinica Chimica Acta, 107(1980) 97 104. *
Lolekha et al., Clinica Chimica Acta, 107(1980) 97-104.
Osberg et al., Clinical Chemistry, vol. 24, No. 7, pp. 1196 1197, 1978. *
Osberg et al., Clinical Chemistry, vol. 24, No. 7, pp. 1196-1197, 1978.
Tagesson et al., Clinical Chemistry, vol. 26, No. 3, 1980, p. 520. *
Wako, Chemical Abstracts, vol. 94, 1981, No. 94: 135591e. *
Wako, Chemical Abstracts, vol. 96, 1982, No. 96: 196193q. *

Cited By (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4818703A (en) * 1985-10-23 1989-04-04 Pizzolante John M Stabilized alkaline picrate reagent for jaffe creatinine determination
US4908676A (en) * 1987-12-18 1990-03-13 Bio-Recovery Systems, Inc. Sensors for dissolved substances in fluids
US5071772A (en) * 1988-02-08 1991-12-10 Raffaele Beli Device for carrying out extemporaneous quantitative diagnostic tests on whole blood
WO1996003648A1 (en) * 1994-07-25 1996-02-08 Professional Clinic Laboratories, Inc. Method of determining renal clearances
US5500372A (en) * 1994-07-25 1996-03-19 Private Clinic Laboratories Inc. Method of determining renal clearances
US5602038A (en) * 1994-07-25 1997-02-11 Private Clinic Laboratories, Inc. Method of determining renal clearances
WO1997027481A1 (en) * 1996-01-25 1997-07-31 Private Clinic Laboratories, Inc. Method of determining renal clearances
US20040023364A1 (en) * 1999-01-15 2004-02-05 Mitchell Owens Lateral flow test strip
US20040228761A1 (en) * 1999-01-15 2004-11-18 Medtox Scientific, Inc. Lateral flow test strip
US20050208677A1 (en) * 1999-01-15 2005-09-22 Medtox Scientific, Inc. Lateral flow test strip
US20070031914A1 (en) * 2005-08-05 2007-02-08 Wei Zhu Devices for analyte assays and methods of use
US20190187146A1 (en) * 2014-02-28 2019-06-20 Nitto Denko Corporation Urinalysis Device And Dry Reagent For Quantitative Urinalysis
US10436792B2 (en) 2014-02-28 2019-10-08 Nitto Denko Corporation Urinalysis device and dry reagent for quantitative urinalysis
US10613097B2 (en) 2014-02-28 2020-04-07 Nitto Denko Corporation Urinalysis device and dry reagent for quantitative urinalysis
US10656160B2 (en) * 2014-02-28 2020-05-19 Nitto Denko Corporation Urinalysis device and dry reagent for quantitative urinalysis

Also Published As

Publication number Publication date
EP0143805A4 (en) 1987-09-02
WO1984003948A1 (en) 1984-10-11
EP0143805A1 (en) 1985-06-12

Similar Documents

Publication Publication Date Title
Yatzidis New method for direct determination of" true" creatinine
EP0432642B1 (en) Reagent composition, methods and kits for quantification of magnesium or calcium and magnesium
US5057435A (en) Reagent and methods for calcium determination
US4626511A (en) Composition for reducing turbidity in samples of biological fluids
US4529708A (en) Assay for the determination of creatinine
US4448889A (en) Fluid analysis
CN108613976B (en) Direct bilirubin detection kit
Datta et al. Interference by IgG paraproteins in the Jaffe method for creatinine determination
CA1089338A (en) Creatinine assay and reagent system
WO1999004258A1 (en) Assay for total and direct bilirubin
JP2603452B2 (en) Method for measuring iron and its reagent
US5866352A (en) Kit for fructosamine determination
JPH11344490A (en) Method for measuring iron without causing interference, and reagent
US4348208A (en) Uric acid assay and reagent system therefor
JP3220378B2 (en) Method and reagent for quantification of total protein
GB2068541A (en) Determination of chloride in serum
JP3713901B2 (en) Method for measuring osteoclast-derived acid phosphatase
US4447544A (en) Method and reagent for determining inorganic phosphate in biological sample
US4800168A (en) Two reagent system for the colorimetric determination of chloride ions in body fluids
US6013465A (en) Method for detection and determination of enzymatic reactions in urine
Deeg et al. A new approach to photometry of glycated hemoglobin in human blood.
Conger et al. Phthalaldehyde microprotein method: Usefulness and potential errors
Stewart et al. Ultramicro Methods in Biochemistry. VII. The Determination of Plasma or Serum Urea Nitrogen, VIII. The Determination of Serum Uric Acid, IX. The Determination of Serum Creatinine
WO1995000843A1 (en) Assay for total bilirubin
EP0597900A1 (en) Colorimetric determination of magnesium ions in fluids

Legal Events

Date Code Title Description
AS Assignment

Owner name: AMERICAN MONITOR CORPORATION, 5425 WEST 84TH ST.,

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST.;ASSIGNOR:STEPHENS, THOMAS W.;REEL/FRAME:004117/0926

Effective date: 19830405

AS Assignment

Owner name: NEUBERGER AND BERMAN (LENDER), 522 FIFTH AVENUE, N

Free format text: SECURITY INTEREST;ASSIGNOR:AMERICAN MONITOR CORPORATION;REEL/FRAME:004610/0276

Effective date: 19860630

REMI Maintenance fee reminder mailed
FPAY Fee payment

Year of fee payment: 4

SULP Surcharge for late payment
AS Assignment

Owner name: AMERICAN MONITOR PARTNERS, 522 FIFTH AVE., NEW YOR

Free format text: SECURITY INTEREST;ASSIGNOR:AM DIAGNOSTICS, A CORP. OF IN;REEL/FRAME:005665/0015

Effective date: 19910114

Owner name: FOOTHILL CAPITAL CORPORATION, A CA. CORP., CALIFOR

Free format text: SECURITY INTEREST;ASSIGNOR:AM DIAGNOSTICS, A CORP. OF IN;REEL/FRAME:005665/0021

Effective date: 19910115

AS Assignment

Owner name: INDIANA DEVELOPMENT FINANCE AUTHORITY, INDIANA

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST.;ASSIGNOR:AM DIAGNOSTICS, INC.;REEL/FRAME:005711/0614

Effective date: 19910131

LAPS Lapse for failure to pay maintenance fees
FP Lapsed due to failure to pay maintenance fee

Effective date: 19930718

STCH Information on status: patent discontinuation

Free format text: PATENT EXPIRED DUE TO NONPAYMENT OF MAINTENANCE FEES UNDER 37 CFR 1.362